Asymmetric Bioreduction of Ethyl 3-Halo-2-oxo-4-phenylbutanoate by Saccharomyces cerevisiae Immobilized in Ca-Alginate Beads with Double Gel Layer Humberto M. S. Milagre, † Cı ´ntia D. F. Milagre, † Paulo J. S. Moran, † Maria Helena A. Santana, ‡ and J. Augusto R. Rodrigues* ,† State UniVersity of Campinas, Institute of Chemistry, CP 6154, CEP 13084-971 Campinas, SP, Brazil, and State UniVersity of Campinas, School of Chemical Engineering, CP 6066, CEP 13081-970 Campinas, SP, Brazil Abstract: The asymmetric bioreduction of ethyl 3-halo-2-oxo-4-phenylbu- tanoate was studied for several microorganisms and especially for Saccharomyces cereWisiae. The highest chemical yield (90%), de (70%) and ee (96-99%) were obtained with S. cereWisiae immobilized in calcium alginate beads with double gel layers, and reaction conditions were optimized by changing matrix of immobilization, concentration of substrate, and feeding with glucose as electron donor. The entrapment of cells with double gel layers was fundamental to achieve high enantio- and diastereoselectivity. Introduction The production of enantiomeric pure compounds is of increasing importance in the fine chemical and pharmaceuti- cal industries in particular. 1 Many of these compounds can be obtained by asymmetric reductions of ketones mediated by Saccharomyces cereVisiae (baker’s yeast). 2 In biocatalytic conversions, 3-7 whole cells are used more often than isolated enzymes since cofactor regeneration is not required for sustained catalytic activity. S. cereVisiae is an economically attractive biocatalyst due to its availability, low cost, ease of handling and disposal, safety for food and pharmaceutical applications, as well as its capacity to catalyze a wide range of stereoselective reductions. 3-8 However, this method has not been considered suitable for large-scale production of chiral alcohol, due to the low concentration of reagents and the long process of isolation of products, since the reactions are generally performed in batch process. Some work with continuous cell-culture systems tried to overcome these disadvantages. 9-12 One alternative is to use immobilized cells for enantioselective reductions of ketones. 13 Several methods for entrapment of living and growing cells have been developed. 14,15 A preparation that provides extremely mild immobilization conditions is the entrapment within ionotropic gels, such as calcium alginate. 16 Entrapment of cells in calcium alginate is the most widely used immobilization technique in the biocatalytic production of chemical compounds. 17-21 Alginate is cheap and readily available, has a high affinity for water, and has the ability to form gels under mild conditions, which are suitable for most cells. It is nontoxic and nonpathogenic, what makes it attractive for applications in the food and the pharmaceutical industries. 20 Several ketone reductions using immobilized cells have been reported in the literature and, in many cases, by S. cereVisiae immobilized in calcium alginate. 13,22-26 Other microorganisms 27,28 as well as other immobilization matrixes * Author for correspondence. E-mail: jaugusto@iqm.unicamp.br. † Institute of Chemistry. ‡ School of Chemical Engineering. (1) Straathof, A. J. J.; Panke, S.; Schmid, A. Curr. Opin. Biotechnol. 2002, 13, 548-556. (2) Nakamura, K.; Yamanaka, R.; Matsuda, T.; Harada, T. Tetrahedron: Asymmetry 2003, 14, 2659-2681. (3) Buque, E. M.; Chin-Joe, I.; Straathof, A. J. J.; Jongejan, J. A.; Heijnen, J. J. Enzyme Microb. Technol. 2002, 31, 656-664. (4) Brzezin ˜ska-Rodak, M.; Z ˙ yman ´czyk-Duda, E.; Kafarski, P.; Lejczak, B. Biotechnol. Prog. 2002, 18, 1287-1291. (5) Chen, J.; Wang, K.; Houng, J.; Lee, S. Biotechnol. Prog. 2002, 18, 1414- 1422. (6) Lourenc ¸ o, E.; Rodrigues, J. A. R.; Moran, P. J. S. J. Mol. Catal. B: Enzym. 2004, 29, 37-40. (7) Filho, E. S. P.; Rodrigues, J. A. R.; Moran, P. J. S. Tetrahedron: Asymmetry 2001, 12, 847-852. (8) Dahl, A. C.; Fjeldberg, M.; Madsen, J. Ø. Tetrahedron Asymmetry 1999, 10, 551-559. (9) Chin-Joe, I.; Haberland, J.; Straathof, A. J. J.; Jongejan, J. A.; Liese, A.; Heijnen, J. J. Enzyme Microb. Technol. 2002, 31, 665-672. (10) Kometani, T.; Yoshii, H.; Matsuno, R. J. Mol. Catal. B: Enzym. 1996, 1, 45-52. (11) Goubet, I.; Maugard, T.; Lamare, S.; Legoy, M. D. Enzyme Microb. Technol. 2002, 31, 425-430. (12) Wendhausen, R., Jr.; Moran, P. J. S.; Joekes, I.; Rodrigues, J. A. R. J. Mol. Catal. B: Enzym. 1998, 5, 69-73. (13) Buque, E. M.; Chin-Joe, I.; Straathof, A. J. J.; Jongejan, J. A.; Heijnen, J. J. Enzyme Microb. Technol. 2002, 31, 656-664. (14) Park, J. K.; Chang, H. N. Biotechnol. AdV. 2000, 18, 303-319. (15) Bickerstaff, G. F. Immobilization of Enzymes and Cells; Humana Press: New Jersey, 1997. (16) Kierstan, M.; Bucke, C. Biotechnol. Bioeng. 1977, 19, 387-397. (17) Smidsrød, O.; Skjåk-Braek, G. Trends Biotechnol. 1990, 8, 71-78. (18) Hannoun, B. J. M.; Stephanopoulos, G. Biotechnol. Bioeng. 1986, 28, 829- 835. (19) Poncelet, D.; Lencki, R.; Beaulieu, C.; Halle, J. P.; Neufeld, R. J.; Fournier, A. Appl. Microbiol. Biotechnol. 1992, 38, 39-45. (20) Martinsen, A.; Storrø, I.; Skjåk-Braek, G. Biotechnol. Bioeng. 1992, 39, 186-194. (21) Hulst, A. C.; Tramper, J.; Van’t Riet, K.; Westerbeek, J. M. M. Biotechnol. Bioeng. 1985, 27, 870-876. (22) Gervais, T. R.; Carta, G.; Gainer, J. L. Biotechnol. Prog. 2003, 19, 389- 395. (23) Griffin, D. R.; Gainer, J. L.; Carta, G. Biotechnol. Prog. 2001, 17, 304- 310. (24) Fadnavis, N. W.; Vadivel, S. K.; Sharfuddin, M.; Bhalerao, U. T. Tetrahedron: Asymmetry 1997, 24, 4003-4006. (25) Naoshima, Y.; Munakata, Y.; Nishiyama, T.; Maeda, J.; Kamezawa, M.; Haramaki, T.; Tachibana, H. World J. Microbiol. Biotechnol. 1991, 7, 219- 224. (26) Talebnia, F.; Niklasson, C.; Taherzadeh, M. J. Biotechnnol. Bioeng. 2005, 90, 345-353. (27) Naoshima, Y.; Akakabe, Y.; Watanabe, F. Agric. Biol. Chem. 1989, 53, 545-547. (28) Martı ´nez-Lagos, F.; Sinisterra, J. V. J. Mol. Catal. B: Enzym. 2005, 36, 1-7. Organic Process Research & Development 2006, 10, 611-617 10.1021/op0502497 CCC: $33.50 © 2006 American Chemical Society Vol. 10, No. 3, 2006 / Organic Process Research & Development • 611 Published on Web 03/10/2006